VARIOUS METHODS OF ANALYSIS OF MDR-1/P-GLYCOPROTEIN IN HUMAN COLON CANCER CELL-LINES
VARIOUS METHODS OF ANALYSIS OF MDR-1/P-GLYCOPROTEIN IN HUMAN COLON CANCER CELL-LINES
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DOI：
10.1093/jnci/84.9.711
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发表时间：
1992-05-06
期刊：
影响因子：
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通讯作者：
FOJO, AT
中科院分区：
文献类型：
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作者：
HERZOG, CE;TREPEL, JB;FOJO, AT
背景：在组织培养系统中已经研究了高水平MDR - 1（也称为PGY1）/ p -糖蛋白（Pgp）介导的多药耐药（MDR）；然而，大多数表达mdr- 1 /Pgp的肿瘤样本的水平要低得多。目的：我们想确定临床观察到的Pgp水平是否可以通过常用的方法检测到，并确定这些水平是否可以通过Pgp拮抗剂逆转MDR。方法：我们研究了耐多药细胞系和亚系，它们的mdr- 1 /Pgp表达水平与临床所见相当。我们通过Northern blot分析、slot blot分析、PCR分析和原位杂交来评估mdr- 1 RNA的表达。我们通过免疫荧光、免疫组织化学、荧光活化细胞分选和免疫印迹分析来评估蛋白表达。通过连续药物暴露过程中细胞的生长情况来确定耐药性和可逆性。结果：在大多数情况下，每种方法都能检测到这些细胞系中存在低水平的mdr- 1 /Pgp，但除PCR法外，其他方法的检测结果均处于灵敏度极限。这些低水平的MDR - 1 /Pgp能够产生MDR，维拉帕米可以拮抗MDR。结论：上述方法均可检测到与临床样品相似的mdr-l/Pgp水平，但PCR方法最灵敏、最可靠。意义：在这些低水平mdr- 1 /Pgp的细胞系中添加维拉帕米的体外致敏表明，临床检测到的水平可能会在体内产生耐药性。
Background: Multidrug resistance (MDR) mediated by high levels of mdr-l (also known as PGY1)/P-glycoprotein (Pgp) has been studied in tissue culture systems; however, most tumor samples which express mdr-l/Pgp have much lower levels. Purpose: We wanted to determine if levels seen clinically could be detected by commonly used methods and to determine if these levels conferred MDR reversible by Pgp antagonists. Methods: We studied multidrug-resistant cell lines and sublines with levels of mdr-l/Pgp expression comparable to those seen clinically. We evaluated the expression of mdr-l RNA by Northern blot analysis, slot blot analysis, polymerase chain reaction (PCR) analysis, and in situ hybridization. We evaluated protein expression by immunofluorescence, immunohistochemistry, fluorescence-activated cell sorting, and immunoblotting analyses. Drug resistance and reversibility were determined by cell growth during continuous drug exposure. Results: In most cases, the low level of mdr-l/Pgp present in these cell lines could be detected by each method, but the assays were at the limit of sensitivity for all methods except the PCR method. These low levels of mdr-l/Pgp are capable of conferring MDR, which can be antagonized by verapamil. Conclusions: Levels of mdr-l/Pgp similar to those found in clinical samples can be detected by each of these methods, but the PCR method was the most sensitive and most reliably quantitative. Implications: In vitro sensitization by the addition of verapamil in cell lines with these low levels of mdr-l/Pgp suggests that clinically detected levels may confer drug resistance in vivo.